1
Hot chicks, cold feet
1 2
Randi Oppermann Moea*, Jon Bohlinb, Andreas Fløc, Guro Vasdald, Solveig Marie Stubsjøene 3
4
aNorwegian University of Life Sciences, Faculty of Veterinary Medicine, Department of 5
Production Animal Clinical Sciences, Animal Welfare Research Group, P.O. Box 8146 dep., 6
N-0033 Oslo, Norway 7
8
b Norwegian Institute of Public Health, Division of Infection Control and Environmental health, 9
Department of infectious disease epidemiology and modeling, Lovisenberggata 8, P.O. Box 10
4404, 0403 Oslo, Norway 11
12
c Norwegian University of Life Sciences, Faculty of Environmental Science and Technology, 13
Department of Mathematical Sciences and Technology, N-1432 Aas, Norway 14
15
dAnimalia, Norwegian Meat and Poultry Research Centre, PO Box 396, Okern, NO-0513 16
Oslo, Norway 17
18
eNorwegian Veterinary Institute, Department of Animal Health and Food Safety, Section for 19
Animal Health, Wildlife and Welfare, P.O. Box 750 Sentrum, N-0106 Oslo, Norway 20
21 22 23
* Corresponding author Randi Oppermann Moe: Tel: +47 67 23 21 17 24
E-mail address: randi.moe@nmbu.no 25
2 26
1. Introduction 27
In recent years, there has been a growing effort to develop scientifically based indicators of 28
emotional states in animals in order to assess their welfare. The subjective components of 29
emotional states cannot be assessed verbally in animals. However, various physiological 30
measurements are used to indirectly detect animal emotions [1,2]. For instance, it has been well 31
documented that acute physical and psychological stress and emotional arousal triggers a 32
sympathetically-mediated cutaneous vasoconstriction causing a rapid drop in skin temperature.
33
This drop is accompanied by a rise in core temperature, followed by a subsequent vasodilatation 34
in order to dissipate excess heat resulting in a post-stressor rise in peripheral temperature. This 35
thermoregulatory response is termed stress-induced hyperthermia, psychogenic fever, or 36
emotional fever, and can be found in mammalian, avian, reptile, and fish species [3-12].
37 38
Infrared thermography (IRT), also known as thermal imaging, is a non-invasive, quantitative 39
diagnostic tool that involves the detection of infrared radiation (heat) emitted from an object 40
[13]. Thermal imaging is used in a broad range of animal studies [14], including studies of 41
stress, emotional arousal, and animal welfare in laying hens [15-19]. For instance, handling 42
stress resulted in an initial surface comb and eye temperature drop within a minute of handling 43
by about 2⁰C and 0.8⁰C, respectively, whilst core temperature rose over a 9-12 min period in 44
laying hens [8,18,19]. Herborn et al. [19] found that the initial stress-induced skin temperature 45
drop (i.e. in comb and wattle) was more pronounced and that the post-stressor rise in 46
temperature was largest in response to the most aversive handling procedure, suggesting that 47
stressor intensity can be quantified by measures of skin temperature alterations in laying hens.
48
Previously, we found evidence that a drop in peripheral temperature may reflect the intensity 49
of emotional arousal rather than its valence, as indicated by a drop in surface comb temperature 50
3
in laying hens during the first minutes of anticipating a palatable food reward [16]. Furthermore, 51
Edgar et al. [15] demonstrated that hens respond to an aversive stimulus directed at their chicks 52
by a drop in eye temperature. These studies suggest that a range of head region temperatures 53
may provide valuable information about stress and emotions in poultry.
54 55
IRT is useful also for the detection of welfare relevant issues not related to stress and emotions 56
in laying hens. For instance, one study showed a positive relationship between IRT records of 57
surface skin temperature and the visual assessment of plumage condition, which indirectly 58
reflects feather pecking behavior in chicken flocks [20]. Furthermore, IRT was useful for the 59
early detection of subclinical leg pathologies (so-called bumble foot) in laying hens [21].
60 61
Taken together, IRT has a great potential to provide valuable information in a variety of animal 62
welfare relevant studies in poultry, ranging from studies of stress and emotions to health related 63
issues [17]. However, although several studies explored temperature in studies of welfare issues 64
in laying hens, less is known about the use of IRT to study welfare in broiler chickens kept for 65
meat production. Leg health problems (e.g. footpad lesions; FPL) are emphasized as important 66
welfare issues in broiler chickens [22], and welfare audits for broilers therefore include the 67
visual inspection of the footpads and scoring of macroscopic appearance of lesion- size and - 68
severity [23]. FPL are associated with inflammatory processes [24,25], which in general are 69
associated with a rise in tissue temperature. Hence, IRT could potentially represent a novel tool 70
for the reliable early detection and/or prediction of subclinical foot pathologies in broiler 71
chickens, as has been suggested for the detection of subclinical bumble foot in laying hens [21].
72 73
However, the use of IRT to study footpad temperatures involves handling and restraint of the 74
birds, which may cause stress and emotional arousal, thus having the potential to affect surface 75
4
temperatures as discussed above. Indeed, foot temperature (in laying hens) may be affected by 76
handling stress: After an initial 6 min drop, the surface temperatures (i.e. interdigital membrane 77
temperature read from a digital infrared thermometer) rose [8]. Although one study showed that 78
immobilization of young small broiler chicks resulted in inconsistent and negligible alterations 79
in abdominal skin temperature [26], there is in general limited knowledge about effects of 80
handling and restraint on surface temperatures assessed from IRT in broiler chickens.
81 82
Therefore, as a basis for the validation of IRT as a future tool for the early detection and/or 83
prediction of subclinical leg pathologies of broiler footpads, this study investigated effects of 84
factors having the potential to affect surface temperature measurements in clinically healthy 85
broiler chickens associated with the assessment of footpad temperatures. The specific aims were 86
to 1) explore effects of manual restraint on footpad temperatures in broiler chickens; 2) 87
investigate footpad temperatures at two different ages, and 3) explore concomitant effects of 88
manual restraint on several surface head region temperatures, in order to gain more knowledge 89
about effects of stress and emotional arousal on surface skin temperatures in broiler chickens.
90 91 92
2. Material and methods 93
94
2.1. Animals and husbandry 95
96
The experiment was carried out at the Institute of Production Animal Clinical Sciences at the 97
Norwegian University of Life Sciences. Twenty broiler chickens (Ross 308) were housed in a 98
pen littered with wood shavings. The chickens were obtained from a commercial producer at 99
5
15 d of age. The birds had ad-lib access to water from a bell drinker and a commercial diet for 100
broilers (KROMAT Kylling 2, Felleskjøpet, Norway) throughout the experiment.
101
2.2. Experimental procedures 102
103
The birds were accustomed to the housing facilities for 15 d before the start of the experiment.
104
Twelve birds were randomly selected for IRT measurements and tested on three test days during 105
a period of seven days, i.e. at 30, 36 (test day 1 and 2; footpad measures) and 37 d of age (test 106
day 3; head region measures). For the footpad measures, each chicken was manually restrained 107
for a total duration of 10 min by a person sitting on a chair. The birds were picked up and gently 108
placed in a position where the ventral side of the feet were pointing upwards towards the thermal 109
camera and with the back leaning against the lap of the handler. The distance between camera 110
and broiler feet was 1m. A cardboard plate covered with aluminium foil to avoid influences of 111
heat emission from the body of the bird and the hands/body of the handler were adjusted and 112
placed on the right leg dorsal to the foot. IRT images of the feet were collected every minute 113
over the 10 min test period (i.e. recordings at 0-9 min). For head temperature recordings, birds 114
were gently picked up and manually handled and restrained in the same position as for footpad 115
images. IRT images of the head were collected at the start and the end of a 10 min time period 116
(i.e. recordings at 0 and 9 min, then the birds were held in an upright position towards a concrete 117
wall, making sure that the distance from the head to the camera was similar (1 m) for all 118
recordings. The experimenters were located in a corner of the same room as the chicken pen 119
and visible to chickens. Birds were sacrificed after the experiment by blunt trauma and cervical 120
dislocation.
121 122
2.3. Infrared thermography 123
124
6
IRT images of the feet and head were collected with a thermal camera (T620bx, FLIR System 125
AB, Danderyd, Sweden). The camera was set to an emissivity of 0.96, and the ambient 126
temperature of the testing room was maintained at 20°C. Relative humidity inside the 127
experimental room was recorded at the beginning and end of every test period. These values 128
were used to allow correction for environmental changes during image analysis. Image analysis 129
software (FLIR ThermaCAM Researcher) was used to determine average surface temperature 130
of the plantar footpad and head (larger anatomical area, see description in Figure 1), and 131
temperatures of the comb base, eye (centre and lateral eye angle), ear, wattles, beak base, and 132
nostril (spot measurements, see description in Figure 2).
133 134
2.4. Statistical analysis 135
136
Linear mixed effects regression was carried out with footpad temperature as the response 137
variable and time in minutes (duration of manual restraint) as a predictor variable. Additionally, 138
sequential testing order and test day of the experiment (i.e. when the chicks were aged 30 and 139
36 d, respectively), were included as predictors to respectively assess putative effects of waiting 140
time before handling, and of age, on footpad temperature. Individual footpad temperature 141
differences, between the chickens nested within age (i.e. test day 1 and 2) with respect to time, 142
were modelled as random slopes:
143
yijkl=Xβ+Zu+εijkl
144
yijkl is the foot-temperature response variable with index i=each sample, j=time in min (0-9), 145
k=individual (i.e. chick) and l=age (i.e. test day 1 or 2). Xβ designates the matrix of fixed effects 146
multiplied with the corresponding parameters to be estimated (β), while the random effects are 147
represented by the matrix Z multiplied with the corresponding parameters (i.e. variances) to be 148
estimated u.
149
7 150
For control, we fitted another regression model with temperatures, recorded at different head 151
regions (the response variable). Temperatures were collected at two different time points (0 and 152
10 min after restraint) and this information was included as a predictor variable to be tested.
153
Sequential testing order was included as a predictor but removed since it was not found to be 154
significant (p=0.66). The individual variance of each chicken was modelled as a random slope 155
nested for each head region with respect to time point:
156
yijkl=Xβ+Zu+εijkl
157
yijkl is the head-temperature response variable with index i=each sample, j=time in min (0 or 9), 158
k=individual (i.e. chick) and l=head region (i.e. beak, wattle, comb, etc.). Again Xβ designates 159
the matrix of fixed effects multiplied with a parameter vector to be estimated β, and the random 160
effects are expressed by the matrix Z also multiplied with parameters to be estimated u. The 161
final model-estimations were carried out using restricted maximum likelihood (REML). Due to 162
the low sample-size we also performed MM-type robust regression [27] with temperature as a 163
response and time point as the explanatory variable for each head region. The quality of the 164
regression models was assessed by examining the residual distribution and by plotting the fitted 165
regression model on the explanatory variables. The Akaike information criterion [28] was used 166
to obtain a quantitative estimate of the model fit. Results from the regression models are 167
reported as mean estimates together with 95% Confidence Intervals (95% CI). The linear mixed 168
effects regression models were fitted using the lme4 package [29]. Degrees of freedom and p- 169
values were computed based on the Satterthwaite method as implemented in the package 170
“lmerTest” [30]. The figures presenting the statistical results were created with the “ggplot2”
171
package [31]. All statistical analyses were performed with the statistical language R [32].
172 173 174
8 3. Results
175 176
One example of a thermal image of a broiler chicken footpad is presented in Figure 1. We found 177
that there was a statistical significant drop (p<0.001) in footpad temperature means during 178
restraining of -0.45 ⁰C 95 % CI (-0.49, -0.41) per minute. Age was also significant (p<0.001) 179
in the sense that temperature rose on average with 1.71 ⁰C 95 % CI (1.04, 2.38) from when the 180
chickens were 30 d of age to 36 d (Figure 3). Sequential testing order of the chickens was also 181
significant (p=0.04) for footpad temperatures with 0.13 ⁰C 95 % CI (0.01, 0.25).
182 183
Examples of thermal images of chicken heads are presented in Figure 2. A significant rise in 184
pooled head region temperature means for t=9 as compared to t=0 (p<0.004) with 0.76 ⁰C 95 185
% CI (0.39, 1.15) was found (Figure 4). We also examined head temperature differences within 186
each specific region of interest using robust regression (Figure 5) and found that only comb 187
base temperature was not statistically significantly different between t=0 and t=9. In all other 188
instances, the temperature rose between the time points. Sequential testing order was not 189
statistically significant for head temperatures (p=0.66).
190 191
9 4. Discussion
192 193
The present study investigated effects of manual restraint and age on footpad and head region 194
temperatures assessed by thermal imaging in healthy broiler chickens. Briefly, manual restraint 195
resulted in a significant temperature drop in footpads and a temperature rise in the head regions, 196
indicative of the thermoregulatory and vasomotor responses previously described as stress- 197
induced hyperthermia or emotional fever [5,6,10,11]. Furthermore, footpad temperatures 198
differed between the two weeks, where birds at 36 d had higher footpad temperatures than at 199
30 d.
200 201
This study is the first to show that manual restraint results in a significant drop in footpad 202
temperatures in broiler chickens. The results are consistent with previous studies in e.g. laying 203
hens, where handling stress and emotional arousal was associated with an initial drop in surface 204
body temperatures, probably due to skin vasoconstriction during the early minutes of stress and 205
arousal [8,16,18,19]. The results suggest that footpad temperatures drop due to cutaneous 206
vasoconstriction in response to manual restraint. In contrast to previous studies of handling 207
stress and foot temperatures in laying hens, where temperature dropped the initial six minutes 208
before it began to rise [8], the footpad temperatures reported here dropped steadily throughout 209
the immobilization procedure (Figure 3). The results may however not be directly comparable, 210
since Cabanac & Aizawa [8] recorded interdigital membrane temperatures as opposed to 211
footpad temperatures recorded here. However, it seems like the footpad temperature began to 212
rise towards the end of immobilization in some of the chickens (Figure 3). Further studies 213
employing a longer restraint duration would be necessary to investigate at what time point 214
broiler footpad temperature begin to rise after the initial drop.
215
10
The order in which the chickens were sequentially sampled affected footpad temperatures: The 216
first chickens restrained had lower temperatures than those restrained last. It could be suggested 217
that human presence during the waiting time and catching process affected thermal responses:
218
All chickens had visual contact with the experimenters throughout the experiment because the 219
pen was in the same room as the experiment was conducted. Furthermore, to capture the 220
chickens, the experimenters entered the pen, which implicated that last chicken caught had been 221
exposed to more catching related disturbances, although none of them showed strong flight 222
responses during capture. This finding may represent a further indication of emotional origin 223
of the temperature alterations found here, and in agreement with studies in group-hosed mice 224
where (colonic) temperature of the last recorded mouse was higher than that of the first mouse 225
in the same cage when recorded sequentially [5]. Thus, duration of manual restraint as well as 226
sequential sampling order need to be taken into account in studies of footpad temperatures in 227
broiler chickens. An effect of sampling order was not found for the head temperature 228
recordings, since head temperatures were only recorded immediately after capture and then 229
after 9 min.
230 231
A rise in most of the head region temperatures was found during the restrain period, which may 232
indicate a rise in deep body temperature and subsequent radiation of excess heat during the 233
course of restraint (Figure 4 and 5). This finding is in agreement with several studies of stress 234
and emotions in homeotherms, and hence in support of an emotional origin of the temperature 235
alterations found here [e.g. 6,11]. For instance, both records of eye temperatures (i.e.
236
temperature recorded in the center of the eye and in the lateral eye angle) rose during 237
immobilization. This finding is in agreement with Edgar et al. [18] who found that even a short 238
period of handling led to a significant rise in eye temperatures. Likewise, a rise in ear and beak 239
base temperature, which are close to the eye region, was detected. Other studies showed an 240
11
initial drop in eye temperatures during handling indicating vasoconstriction (5 s) before the 241
temperatures rose to levels significantly higher than baseline temperatures to dissipate heat 242
[18,19]. A similar initial drop was not detected here, probably due to the fact that chickens were 243
exposed to capture and handling before the initial temperatures were measured, as opposed to 244
IRT measures of baseline temperatures reported in undisturbed hens [18,19], and no further 245
measurements were undertaken before the last measurement at 9 min. Earlier studies showed 246
that arousal was associated with raised brain temperatures in chickens [33]. Thus, it could be 247
speculated that the close proximity between brain and eye may have influenced eye 248
temperatures recorded here. Furthermore, eye temperature has been suggested to represent a 249
good indicator of core temperature [34]. Although core temperature was not recorded here, it is 250
likely that eye temperature rise indicate a rise in core temperature due to restraint as reported in 251
previous studies [8,18,35]. A rise in nostril temperatures during immobilization may further 252
indicate that core temperature actually had risen, and that excess heat was dissipated by 253
exhalation through the nostrils in addition to a peripheral vasodilatation in the head regions.
254
Furthermore, from the thermal images it was observed that some of the chickens had a slightly 255
open beak at the last recording (9 min), which may indicate that they panted to dissipate heat 256
(see Figure 2b).
257 258
The rise in head temperatures during restraint were in agreement with previous studies [18].
259
The wattles, which together with the comb have an important role in temperature regulation 260
due to their high density of arteriovenous-anastomoses [36-38], showed a rise in temperature 261
due to restraint, in agreement with previous studies [18,19]. However, in contrast to these 262
studies, the comb base temperature was not significantly affected. This lack of effect is most 263
likely a result of the very small size of the comb at this early age and the difficulty to precisely 264
identify the comb base on the thermal image (Figure 2). Thus, studies of stress and emotions in 265
12
broiler chickens could benefit from replacing spot measures of skin areas of specific interest 266
with average temperatures based on recordings of larger skin areas in the head region. Indeed, 267
head average as well as maximum temperatures (which were recorded on a larger area, in 268
contrast to the specific region spot measurements) were clearly affected by manual restraint, 269
and the individual head region temperatures did not give additional information about emotional 270
arousal during restraint (Figure 5). Therefore, average or maximum head temperature could be 271
employed as a feasible measure of emotional state in future studies of stress and emotions in 272
broiler chickens.
273 274
We found evidence that chickens at 36 d consequently had higher footpad temperatures at each 275
recorded point than at 30 days of age (Figure 3). This could be a result of age effects. It was 276
previously found that surface skin temperature measures in the abdominal area drops as a 277
function of age in broiler chickens [39], and the results therefore stand in contrast to previous 278
findings. It is not clear why age affected footpad temperatures. It could be speculated that age 279
dependent anatomical and/or histological alterations, and alterations in circulatory or 280
thermoregulatory capacity due to age, could explain the results. On the other hand, Herborn et 281
al. [19] found that the most aversive handling procedures resulted in higher temperatures. Thus, 282
if repeated restraint (i.e. measurements week 2) was experienced as more aversive due to a 283
conditioned response than restraint in the first week, then it could be suggested that the results 284
reflect an effect of repeated handling and restraint. Indeed, a study in rats showed that repeated 285
(colonic) temperature measurements resulted in a conditioned temperature rise the second 286
week, whereas a gradual habituation and temperature drop was found at later measurements.
287
On the other hand, rectal temperature in mice handled for rectal temperature measurement and 288
reused after 7 or 14 days did not differ from day 1, implying that mice can be reused in studies 289
of stress-induced hyperthermia [5]. Chickens here served as their own controls, and it is 290
13
therefore not possible to draw conclusions whether the higher temperatures recorded the second 291
week were an effect of age dependent alterations or conditioning/habituation to the handling 292
procedure.
293 294
The effects of handling, restraint and sampling order on footpad temperatures could have 295
clinical relevance in veterinary IRT scanning for footpad lesions. If the magnitude of a 296
temperature rise due to e.g. subclinical lesions is low, and if the duration of veterinary 297
procedures involved is prolonged, it could be that emotion-induced confounding temperature 298
effects in IRT measurements of lesions could affect conclusions of such studies. Clearly, 299
duration of capture and restraint as well as sampling order should be included in future 300
experimental protocols in studies of surface temperature of broiler footpads. Further studies 301
will be needed to address how much of a potential inflammation-induced temperature increase 302
that could theoretically be masked by the emotion-induced cooling of feet.
303 304
While the statistical models employed in the present study exhibited a good fit to the data, the 305
number of sample points, especially for the head-temperature measurements, may obscure 306
certain effects due to variance problems associates with low-sample sizes. This was most 307
pronounced with respect to testing order as a weak effect was observed for foot temperatures, 308
but not for head temperatures. On the other hand, the weakness of this effect with regards to 309
footpad temperatures calls for caution, although there are several reasons arguing for such an 310
effect. Nevertheless, our result could indicate interesting avenues for future research on stress 311
and emotions in broiler chickens.
312 313 314 315
14 5. Conclusions
316 317
This study is the first to demonstrate that footpad temperatures drop whereas head region 318
temperatures rise in response to manual restraint duration in broiler chickens, consistent with 319
body temperatures alterations due to stress and emotional arousal termed stress-induced 320
hyperthermia or emotional fever. Furthermore, footpad temperature differed between 30 and 36 321
d of age, but it is impossible to draw conclusions whether this effect was caused by age or by 322
previous experience (i.e. due to habituation or fear-conditioning). Furthermore, sequential 323
sampling order affected temperature. Thus, one needs to take into account several factors such 324
as the duration of handling and restraint as well as the chickens age, previous experience of the 325
birds and sequential sampling order when using IRT technology in future studies aimed at the 326
early detection and/or prediction of subclinical footpad pathologies in broiler chickens.
327 328 329
Acknowledgements 330
331
We thank the staff at the Department of Production Animal Clinical Sciences at the Norwegian 332
University of Life Sciences for taking care of the chickens. Sverre Futsæter is greatly 333
acknowledged for excellent technical assistance with analyzing the thermal images. This project 334
was funded by the Norwegian Research Council [NFR-project no. 234191], the Foundation for 335
Research Levy on Agricultural Products, the Agricultural Agreement Research Fund, and 336
Animalia — Norwegian Meat & Poultry Research Centre. Jon Bohlin was funded by the 337
Norwegian Institute of Public Health.
338 339 340
15 Figure 1: Thermal image of a footpad
341 342
The figure shows one example of a thermal image of one individual broiler chickens’ footpad.
343
The circle illustrates the anatomical area that was measured. A circle was created within the 344
software to cover as much as possible of the footpad, without covering areas outside of the 345
footpad.
346 347
348 349
16
Figure 2: Description of the anatomical location for the different areas and measurement 350
spots included in head temperature recordings.
351 352
The figure shows examples of thermal images of broiler chicken heads, and the anatomical 353
location of the areas and measurement spots that were included in the analyses. Figure 2a shows 354
a typical image that depicts the anatomical area where “Head average” and “Head max”
355
temperatures were measured. Here, a circle was created within the software to cover as much 356
as possible of the head, without covering areas outside the head. Figure 2b shows a typical 357
example of the anatomical areas measured as specific measurement spots; Sp1: Eye angle 358
(lateral eye angle, which includes vascularized areas), Sp2: Eye center (middle of the eye), Sp3:
359
Nostril, Sp4: Comb base, Sp5: Wattle, Sp6: Ear, and Sp7: Beak base.
360
361
2a 2b
362
17
Figure 3: Footpad temperatures in twelve individual broiler chickens during 10 min. manual 363
restraint, at 30 and 36 d of age.
364
365 366 367
The figure shows recorded footpad temperatures (vertical axis), in degrees of Celsius, plotted 368
against time in minutes (horizontal axis), together with regression model estimates (black 369
points). Each panel, from 1-12 represent each individual chicken with footpad temperatures 370
taken at weeks 1 and 2; i.e. 30 and 36 d of age (red and blue lines).
371 372
18
Figure 4: Head region temperatures (pooled) in response to manual restraint.
373
374 375
The figure shows a box plot of pooled head region temperature measurements (black points) 376
plotted against two time points (i.e. 0 and 9 min). The red points are temperatures estimated by 377
the mixed effects regression model.
378 379 380 381
19
Figure 5: Head region temperatures in response to manual restraint.
382 383
384 385
The box-plots show head temperature measurements (y-axis) with respect to time (0 and 9 min, 386
x-axis). Each panel corresponds to a separate head region from which temperatures were 387
measured. The stars indicate statistical significance at the p<0.05 level (*) p<0.01 level (**) 388
and p<0.001 level (***).
389 390 391 392
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